Preservation of biologically active substances



y 7, 1940- E. w. FLOSDORF 2.199.817

PRESERVATION 0F BIOLOGICALLY ACTIVE SUBSTANCES Original Filed Dec. 12,1935 lNVE/TEOR w M ATTORNEYS Patented May 7, 1940 UNITED STATES PATENTOFFICE PRESERVATION 0F BIOLOGICALLY A SUBSTANCES Earl W. Flosdorl',Ardmore, Pa., assignor to The Trustees of the University ofPennsylvania, Philadelphia, Pa., a corporation of Pennsylvania Originalapplication December 12, 1935, Serial No.

Divided and this application October 17, 1936, Serial No. 106,105

3 Claims.

vacuum, the container being provided with a.

novel rubber stopper which has a passage therethrough and an integralrubber tubular extension of said passage, the container being sealed byclamping off this rubber extension afterthe biologically activesubstance has been desiccated and while a vacuum is maintained withinthe container, by means of a metal clamp.

This application is a division of my prior application Serial No.54,148, filed December 12, 1935, in which is described a process for thepreservation of biological substances in which the biological substancesin a liquid state are introduced into a final container adapted for thedistribution of the product, and are rapidly and thoroughly frozentherein, and then are dehydrated by the use of a high vacuum, andsealed, after dehydration, in the containers while under a high vacuumand without destroying the vacuum under which the dehydration waseffected, so that a desiccated product, sealed under the originalvacuum, with maintenance of asepsis and prevention of deterioration andcontamination is obtained. In the process therein described, a largenumber of small market containers may be simultaneously processed, andvarious modifications of suitable and novel containers, with thedesiccated material sealed in them under the original vacuum, aredescribed. Some of the containers are provided with a-rubber stopperprovided with a passage through which a glass tube may be passed, sothat the application of the high vacuum, and the dehydration of theproduct, may be, carried out by attaching the glass tube to a suitablemanifold; in other suitable forms of containers, there is provided arubber stopper having a passage, with a tubular rubber extension of thepassage integral with the stopper, so that this tubular extension may beconnected, as by means of a glass ube, to a vacuum manifold to permitthe application of a high vacuum and the dehydration of the frozenmaterial.

In the present application, I claim the improved containers, providedwith such a rubber stopper, containing desiccated biological substances,sealed under a high vacuum by means of a metal clamp on the tubularrubber extension.

The stoppers which are used are advantageously provided with apassage,'in addition to the passage continuous with the tubularextension, extending part way through the stopper, which provides aperforable membrane through which sterile water can be readilyintroduced to restore the desiccated product to a liquid conditionwithout destroying the vacuum until the water has been so introduced.The restoration of the product to a liquid state in this way enables theproduction of a. restored product with assurance that the originalproduct has not been contaminated A in any way from the time theoriginal liquid was introduced into the final container until it isultimately restored to a liquid state. Asepsis is thus maintainable fromthe time the original serum or other product is inserted in thecontainer until it isrestored by the addition of sterile water.Furthermore, by the introduction of the the sterile water while a vacuumis maintained within the container, the dissolution of the desiccatedmaterial is more rapid and complete than if the water is introducedafter air has been permitted to enter the container, as the air tends topermeate the desiccated material, which is very porous, and to slow upor prevent the penetration of the material by the water.

It is important, in order to produce, in a final market containersuitable for distribution and use, amounts of desiccated materialsuitable for clinical use as full individual doses or multiples thereof,and to enable the material to be advantageously processed, to maintain aproper ratio between the volume of the original serum or other materialin the container and the surface area of the frozen material in contactWith the container and the area of the interior surface from which theevaporation or sublimation of ice takes place. At the end of theprocess, the sealed container'contains the desiccated product in anundisintegrated state and with practically the volume of the originalliquid, and, in general, with the same or a similar ratio of volume andsurface area, although the final product, because of its light andporous nature, has enormous interior surfaces.

It' is also important, in order to produce clinically usable amounts ofthe product in the final containers, to provide exhaust tubes, forremoving the water vapor from the containers, which are properlyproportioned, and which are sealed at the end of the process to hold theoriginal vacuum.

The invention will be further described in connection with specificillustrations.

The containers.--The containers or receptacles which are used are of asize and shape which adapt them for containing the original liquidmaterial, for permitting freezing of this liquid in the containers andfor desiccation of the frozen material, and these containers are also toserve as'the final containers for storage of the dry product and forrestoring the desiccated material to its original liquid state. Thecontainers should be of asize and shape which adapts them for use bothas the containers of the material during the process, and as thecontainers of the final product.

In size, the containers should in general be at least twice the size ofthe liquid serum or other material to be processed therein, so that thevolume of material put into a container will not exceed about one-halfthe capacity of the container.

While containers as small as 2 to 5 ml. capacity are suitable andconvenient for the preservation of materials in small amounts, in thecase of such materials as virus suspensions or bacterial cultures,larger containers will in general be used, for example, up to containersof 50 ml. capacity for amounts of serum and similar products up to about25 ml. in amount, which is the unit of such material generally processedin a single container for clinical use.

Larger containers can, however, be used, when desired, such as formultiple unit amounts of the product. Containers of about 200 ml.capacity, or about 8 ounces, are suitable for processing and preservingamounts of materials up to around m1. In certain of its aspects, theinvention includes the use of containers for desiccating volumes of asmuch as 100 milliliters to 1 liter or more, in a container ofcorrespondingly increased size, since even such large containers can beeffectively sealed under the original vacuum by the rubber tube sealingmethod of the present invention. But, in general, for clinically usableamounts of the biological materials, the individual unit will not exceedaround 20 to 25 ml., and the containers need not, in general, exceedabout 50 ml. capacity, when properly proportioned. For certainmaterials,'however, such as breast milk, containers of around 200 ml. or8 ounces capacity are suitable. Certain nursing bottles of the standardclean easy type, when constructed with a suitably shaped andproportioned neck, are suitable for use.

The following table shows typical container sizes, with an indication ofthe approximate container volume, the maximum volume of serum or othermaterials to be processed,-and to be contained in the container, thebody length, diameter and wall thickness, and the length and diameter ofthe necks of the containers to adapt them for the rubber stopperclosures by which an effective vacuum is to be maintained in the finalcontainers.

Table 1 Neck inside A prox- Body ignite Body Wall Neck diameter gggserum length 59 length 11 volume mlume eter :31 5 3 13 ml. 'ml. mm. mm.'mm. mm. mm. mm 1 00 50 35 2. 0 15 15. 2 13. 7 50 25 110 28 1. 5 15 15.2 13. 7 a 2 a t it :2 a; e; l.

6 3 45 15. 5 1. 0 15 15. 2 l3. 7 2 1 Sphere 22 1. 0 l0 5. 4 4. 5 2 1 2216 1. 0 l1 7. 8 6. 8

From this table it will be noted that the size and shape of the neck,which is to receive the rubber stopper, is the same for containers ofconsiderable variations in volume. This is advantageous and it enables astandard rubber In general, the ratio of the interior serum evapostopperto be used, and one which is of a size which enables the container to berapidly evacuated through an opening of suitable size extendingtherethrough.

.. The shape and proportions of the containers are also important inprocessing and producing clinically useful amounts of the serum, etc.,in the final sealed containers. For processing of the material in aneffective and reliable manner, the size and shape of the containers areadvantageously such that a layer of frozen material on the side of thecylindrical container while in a horizontal position, after the initialliquid is frozen, is not less than 3 mm. in thickness at the verticaldiameter nor more than 15 mm.; and also such, as pointed out, that thevolume of the frozen material does not exceed about one-half the volumeof the container. The proper shape and proportions of the container alsoenable a desirable ratio to be obtained between the volume of the frozenmaterial, during processing, the area of the frozen material in contactwith the walls of the container, and the interior area of the frozenmaterial which provides the evaporating surface during the dehydrationof the frozen material. The final product, in the sealed evacuatedcontainer, will have in general the same volume and shape as that of thefrozen product, although the final product will be a porous solid.

30 rating surface to the exterior surface of the frozen serum in contactwith the walls of the container should be such that the evaporatingsurface has an area of at least about one-half that of the area of thefrozen material in contact with the walls of the container. In general,also, there shouldbe a minimum of about one to two square centimeters ofinternal evaporating surface per milliliter of serum frozen. Thesevolume-area relations, which are important during the production of theproduct, are retained in general by the final product, even though it isno longer in a frozen condition, but instead in a light porous state.

The container stoppers-For high vacuum tightness, a great compression ofthe rubber is necessary. This is obtained by tapering both the neck ofthe container and the stopper, and by lubricating the stopper before itis inserted, so that there is a maximum of compression on the entirelength of the neck surface. The upper limit of compression is that whichwill not squeeze the holes to too small a diameter and which will notcause undue distortion of the stopper because of unequal stresses overits length. This procedure also tends to reduce the porosity of therubber. In inserting the stoppers in the containers, they should befirst lubricated'with sterile distilled water or dilute antisepticsolution, since a stopper which does not require lubrication would notin general be sufliciently tight. The rubber stock of which the stoppersare made should be a fairly pure gum stock containing some fillercontainers such aare by Table'l above are given in the following table:

The exhaust tubes-The exhaust tubes of the rubber stoppers, which aremolded integrally with the rubber stoppers must be of such characterthat they can be effectively sealed by means of a metal clamp with avacuum-tight seal, and also must be sufiiciently large to permit thenecessary escape of water vapor during the dehydration of the frozenproduct. The first rubber stopper in the above table has an integralrubber tube extension about 32.n1m. long, 9.5 mm. outside diameter, and6.5 mm. inside diameter, the inside of the tube furnishing and formingan extension of the exhaust hole in the stopper itself. For containersup to about 25 cc. liquid content, a tube of about 3 mm. inside diameteris the minimum size that is satisfactory. For containers from about 25cc. up to about cc. content of frozen material, an exhaust tube having aminimum inner diameter of from 4.0 to 5.0 mm. is necessary.

Stoppers of this type may be used not only with small market containersof the kind described, but also with much larger containers, since tubesof larger diameter and of sufficient wall thickness can readily be madeof soft rubber to withstand the high vacuum and to permit suiiicientcompression to seal the containers. For example, in sealing a containercontaining up to 20 or 25 litres of material, a rubber tube may be usedwhich is as much as 2 inches in diameter internally, and with a wallthickness of about 1 inch. In this case, strong metal clamps arerequired for making a vacuum-tight seal, for example, steel bars whichclamp together with a space between them of 1%.11101188 or even 1 inch,so that the soft rubber of the tubes is very highly compressed by theclamps. Rubber of a relatively soft variety should be used for suchtubes.

With the small final containers, for marketing and distribution, thecontainer may be finally sealed, at the end of the process, bycompressing it sufiiciently between parallel metal clamping surfacesunder a sufi'icient pressure to hold a permanent vacuum seal on. thetubing. The metal clamp must be of sufiicient strength and rigidity tohold the rubber permanently compressed.

The invention will be further described in connection with theaccompanying drawing, which illustrates in a somewhat conventional anddiagrammatic manner, a suitable rubber stopper, and a container providedwith such a rubber stopper as attached to a vacuum manifold, and acontainer, containing desiccated material, as finally sealed.

In the drawing,

Fig. 1 is a sectional view of a suitable rubber s p r;

Fig. 2 is a sectional view of a container provided with the rubberstopper of Fig. l, as attached to a vacuum manifold; and

Fig. 3 ,is a perspective view of a final sealed container. 0

The rubber stopper illustrated in Fig. 1 is tapered, as shown at ill, toprovide compression all along the surface of contact between it and Jthe container,'and is provided with a tubular passage II which iscontinuous with the passage of the integrally formed tubular extensionIt, to form a suitable exhaust passage. The stopper is also providedwith another passage ll ex-i tending part way through it, leaving a thinsection I! through which a hypodermic needlev can be inserted when it isdesired to restore theproduct by the addition of water. A slightdepression I6 may be provided above this thin section, or 15 membrane,to facilitate locating it. In the container shown in Fig. 2, thereisprovided a neck It, which is tapered as is the rubber stopper, andwith, a rubber stopper such as illustrated in Fig. 1 inserted intheneck. Before in- 20 setting this stopper, a liquid'biologically activesubstance is introduced into the container, this substance beingsubsequently frozen and dehydrated by the application of a vacuum. Ametal ring I! is placed over the tubular extension it of 25 the rubberstopper, and a glass tube It, which is L-shaped, is inserted in the endof the tubular extension, to permit attaching the container to a vacuummanifold. After the liquid material is introduced into the container,and the stopper 0 and glass tube are attached thereto, the material inthe container is frozen by immersing the container, preferably in ahoriiiontal position, in a suitable freezing mixture, e. g. a freezingmixture of a temperature of 70 C. or lower, and the 35' container isthen attached to a vacuum manifold by means of the L-shaped glass tube,and the material in the container is desiccated or dehydrated by thevacuum.

After the dehydrationhas been completed, the 4.0 metal ring I! iscompressed between parallel clamping surfaces,'so that it forms a metalclamp on the rubber tubular extension as shown at [9 in Fig. 3, and therubber tubular extension is then cut off a little above this metalclamp, giving 45 the final container illustrated in Fig. 3 with thedehydrated or desiccated material within it sealed under the originalhigh vacuum, aseptically, and free from contamination.

. The container can be further protected by coat- 59 ing or covering theentire exposed rubber portion with a liquid sealing cement, whichretains its flexibility and imperviousness when dry, and aids inpreventing deterioration of the rubber, as well as in preserving thevacuum within the container. 55

I claim:

1. As a new article of manufacture, a. sealed evacuated container,containing asolid, dry or lyophilic biologically active substance, saidcontainer having a neck and a tightly fitting rubber 5i} stopper closurefor said neck to maintain the vacuum therein, said closure for said neckhaving 'an opening therethrough and an integral tubular extension,sealed by a clamp with a vacuumtight seal, said closure permitting thepassage of $5 a tubular member for the addition of water to restore theproduct to a liquid state without de-' stroying the vacuum, and thesolid, dry or lyophilic biological substance therein having a volumecorresponding generally to the volume of 70 the initial liquid substanceand occupying around half or somewhat less than half of the total volumeof the container.

2. As a new article of manufacture, an evacuated container sealed underthe original vacuum 15 used in dehydratingjhe biologically activesubstance containing a solid, dry or lyophilic biologically activesubstance, said container having a neck and a tightly fitting rubberstopper closure for'said neck to maintain the vacuum therein, saidclosure for said neck having an opening therethrough and an integraltubular extension, sealed by a clamp with a vacuum-tight seal, saidclosure permitting the passage of a tubular memher for the addition ofwater to restore the product to. a liquid state without destroying thevacuum, and the solid, dry or lyophilic biological substance thereinhaving a volume corresponding half the initial thickness of the walls tomaintain 10 a high vacuum in the container.

EARL W. FLOSDORF.

